[[Image:Oxidative_burst_system_design.png|thumb|center|600px|Figure 3 - System design for the oxidative burst.]]

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The entire system will be engineered onto a high copy plasmid in E. coli as seen in Figure 3. Promoters are shown as bent, thin arrows, genes as thick arrows, ribosome binding sites as blue ovals, and a double transcriptional terminator as two red circles. Initially, the system will function independently, with luxR under a constitutive promoter. Later, luxR will be put under the control of a recombinase system that will only turn on the pathway randomly in a subset of cells. This will be one of three fates the master strain will be able to differenciate into. For more information, visit the [[/Project/Population_Variation|population variation]] page.

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An ideal system would behave as outlined in Figure 4. It shows the relative abundances of the proteins and molecules used in the system. It is only meant to show if a particular species is relatively high or low, not its absolute concentration.

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[[Image:Oxidative_burst_system_response.png|thumb|center|600px|Figure 4 - Ideal system response for the engineered E. coli after being exposed to AHL.]]

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===Current Progress===

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*'''Characterization of LuxR receiver:''' The luxR receiver (F2622)is able to turn on transcription of GFP reporter in the presense of acyl-homoserine lactone (AHL). The part begins to switch on around 100pM AHL and has a 43x fold increase in GFP flourescence between the induced (10nM AHL) and uninduced states. Although saturation of the luxR switch was not observed at 10nM AHL, it is difficult to characterize behavior at 100 nM or 1000 nM AHL because the cells grow very poorly. Presumably, this is due to a high protein load. Therefore, saturation of the luxR reciever likely occurs between 100 and 1000 nM AHL. Flow cytometry data did show the receiver to be leaky, as compared to a control without a GFP reporter, even in the uninduced state. In the final construct, the GFP reporter will be replaced by the oxidase. Thus this data shows our engineered cells will be able to activate the appropriate gene in response to detecting an invading pathogen, although there will likely be a basal level of oxidase expression. All cells (DH10B) were grown in M9 + 2% glucose prior to flow cytometry.

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*'''Characterization of TetR inverter:''' Besides turning on an oxidase, the engineered E. coli need to turn off transcription of their protective catalase. In the uninduced state, the catalase serves as a buffer against leaky oxidase expression. Flow cytometry data shows that when the tetR inverter (Q04400) is cloned downstream of the luxR receiver, it is able to repress GFP expression to that of the negative control (no GFP reporter). This bods very well as catalase will need to be tightly repressed in order to allow H<sub>2</sub>O<sub>2</sub> to accumulate to bactericidal concentrations. The addition of an LAA tag to the catalase should not only ensure fast clearance of the catalase one the cell is induced, but also further reduced any possible leaky expression.

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*'''Galactose oxidase characterization:''' Galactose oxidase has proven to be a difficult enzyme to get to function ''in vivo''. Every ''in vitro'' test (essentially just lysing the cells in a sodium phosphate buffer containing CuSO<sub>4</sub> and testing the soluble fraction) has shown galactose oxidase to be consistently very active. However cells expressing galactose oxidase fail to produce detectable amounts of H2O2 in the supernatant. This is true even of JI377 cells (DE katE, katG, ahp) which cannot scavenge hydrogen peroxide. The leading hypothesis for this inactivity is that galactose oxidase doesn't have sufficient access to its cofactor, Cu<sup>2+</sup>. Cells grown with copper, thoroughly washed, and then lysed show a great reduction in the activity of galactose oxidase. For this reason, we are looking into the alternative oxidase, pyruvate oxidase.

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*'''Pyruvate oxidase:''' Pyruvate oxidase (''spxB'') from ''Streptococcus pnumoniea'' (R6) is being explored for its ability to produce H<sub>2</sub>O<sub>2</sub> ''in vivo''. This enzyme is responsible for the hydrogen peroxide that this pathogen naturally produces when it infects the lungs of humans. The most attractive part of this enzyme it that there should be no transport issues for its substrait (pyruvate) into the cell. Pyruvate oxidase is currently being tested in ''E. coli'' in a catalase proficient strain (DH10B) and a catalase deficient strain (JI377), either under the control of a strong constitutive promoter (J23100.B0034) or an AHL inducible promoter (F2622.B0034). The first bit of good news is that catalase deficient cells cannot grow when constitutively expressing pyruvate oxidase, while catalse proficient cells can. This suggests the toxicity is mediated by H<sub>2</sub>O<sub>2</sub> rather than high protein loads or the toxicity of pyruvate oxidase.

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===Cloning Progress===

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* 8/5/08

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**Good by colony PCR: Pyruvate oxidase in front of a double terminator (spxB.B0015)

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**Successful SDM of katG and katGssrA to remove EcoRI site.

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*8/11/08

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**Pyruvate oxidase is under the control of a strong constitive promoter (J23100.B0034) and an AHL inducible promoter (F2622.B0034)

Current revision

Contents

Oxidative Burst

The General Idea

In order to help guard against infections of the gut, we wish to engineer a strain of E. coli capable of killing bacterial pathogens. White blood cells (neutrophils in particular) are already very efficient at killing bacteria. They do this by engulfing the bacteria and exposing it to a bombardment of reactive oxygen species. These include superoxide, hydrogen peroxide and hydrochlorous acid. The reactive oxygen species kill the bacteria by shredding any biological molecule they come in contact with by way of their potent oxidizing properties. However neutrophils are not able to migrate to the large intestinal lumen where pathogens can reside. Because bacteria are well adapted to live in the gut, this project’s goal is to engineer a strain of E. coli to seek out and kill invading bacterial pathogens by means of a sudden burst of hydrogen peroxide.

Detection

Figure 1 - Quorum sensing in gram negative bacteria

Bacteria are able to communicate between individuals of the same species by way of quorum sensing. Small molecules serve as the signal between individual cells. Gram negative bacteria use acylhomoserine lactones (AHL), which can freely diffuse across the cell membrane. The quorum sensing machinery relies in two enzymes, LuxI, an AHL producer, and LuxR, an AHL-dependent transcriptional activator. Figure 1 illustrates how the system works. In isolation, each bacterium constitutively produces a small amount of AHL, which quickly diffuses into the surrounds. If other bacteria of the same species are also nearby, the AHL will diffuse across their membrane where it will bind to LuxR. LuxR activates transcription of several genes, including luxI. A positive feedback loop is created, in which more AHL induces more LuxI, which in turn produces more AHL. Each species of gram negative bacteria produces a unique AHL, requiring unique LuxI and LuxR proteins, and so avoids crosstalk between species. A group of bacteria can thus toggle between an “off” state and an “on” state by using quorum sensing.

Figure 2 - Detection scheme used by our engineered E. coli

Our engineered strain will not participate directly in quorum sensing, but instead will eavesdrop on the conversation. It will be engineered to constitutively express a LuxR able to detect a species AHL. In this way, our engineered strain will be able to be tuned to specifically respond to a variety of bacterial pathogens. Once the AHL is bound, LuxR will activate a set of genes which will lead to the overproduction of hydrogen peroxide, killing the invading cell.

Response

After sensing the presence of an invading pathogen, we want to engineer our E. coli to produce lethal amounts of hydrogen peroxide relatively quickly. We are not concerned with having the engineered E. coli survive either, as it is reasonable to assume that there are “unactivated” cells far away from the pathogen that could sustain the population. After LuxR binds AHL, it will activate transcription of an oxidase (an enzyme that produces hydrogen peroxide). There are many enzymes that can produce hydrogen peroxide in a stoichiometric ratio. The enzyme currently being used is galactose oxidase, as it has already undergone directed evolution to optimize its thermo stability and has been shown to be highly active in vitro. Other enzymes being considered are pyruvate oxidase and aldehyde oxidase.

Even though our engineered cells will eventually die from their oxidative burst, we want them to survive long enough to produce large amounts of the oxidase so they can produce large amounts of hydrogen peroxide. If the cells were left to produce hydrogen peroxide without any protection, they would produce just enough to be cytotoxic and then fissle, killing only themselves but not much else. To avoid this problem, an E. coli catalase will be constitutively expressed, and then turned off shortly after the oxidase is being expreseed. We’re accomplishing this by putting katE (on of two E. coli catalase genes) behind the tetR sensitive promoter (tetR P) and having tetR co-transciptionally expressed with the oxidase. The time it takes for tetR to accumulate in the cell provides the delay in repressing katE expression. To ensure katE is rapidly cleared from the cells, it has a C-terminal ssrA degradation tag, which should reduce the protein’s half life to the order of minutes. In this way, the cell can be temporarily protected from hydrogen peroxide, but large amounts can accumulate before the substrate is exhausted.The final strain will have deletions of both catalases, ensuring no interference.

System Design

Figure 3 - System design for the oxidative burst.

The entire system will be engineered onto a high copy plasmid in E. coli as seen in Figure 3. Promoters are shown as bent, thin arrows, genes as thick arrows, ribosome binding sites as blue ovals, and a double transcriptional terminator as two red circles. Initially, the system will function independently, with luxR under a constitutive promoter. Later, luxR will be put under the control of a recombinase system that will only turn on the pathway randomly in a subset of cells. This will be one of three fates the master strain will be able to differenciate into. For more information, visit the population variation page.

An ideal system would behave as outlined in Figure 4. It shows the relative abundances of the proteins and molecules used in the system. It is only meant to show if a particular species is relatively high or low, not its absolute concentration.

Figure 4 - Ideal system response for the engineered E. coli after being exposed to AHL.

Current Progress

Characterization of LuxR receiver: The luxR receiver (F2622)is able to turn on transcription of GFP reporter in the presense of acyl-homoserine lactone (AHL). The part begins to switch on around 100pM AHL and has a 43x fold increase in GFP flourescence between the induced (10nM AHL) and uninduced states. Although saturation of the luxR switch was not observed at 10nM AHL, it is difficult to characterize behavior at 100 nM or 1000 nM AHL because the cells grow very poorly. Presumably, this is due to a high protein load. Therefore, saturation of the luxR reciever likely occurs between 100 and 1000 nM AHL. Flow cytometry data did show the receiver to be leaky, as compared to a control without a GFP reporter, even in the uninduced state. In the final construct, the GFP reporter will be replaced by the oxidase. Thus this data shows our engineered cells will be able to activate the appropriate gene in response to detecting an invading pathogen, although there will likely be a basal level of oxidase expression. All cells (DH10B) were grown in M9 + 2% glucose prior to flow cytometry.

Characterization of TetR inverter: Besides turning on an oxidase, the engineered E. coli need to turn off transcription of their protective catalase. In the uninduced state, the catalase serves as a buffer against leaky oxidase expression. Flow cytometry data shows that when the tetR inverter (Q04400) is cloned downstream of the luxR receiver, it is able to repress GFP expression to that of the negative control (no GFP reporter). This bods very well as catalase will need to be tightly repressed in order to allow H2O2 to accumulate to bactericidal concentrations. The addition of an LAA tag to the catalase should not only ensure fast clearance of the catalase one the cell is induced, but also further reduced any possible leaky expression.

Galactose oxidase characterization: Galactose oxidase has proven to be a difficult enzyme to get to function in vivo. Every in vitro test (essentially just lysing the cells in a sodium phosphate buffer containing CuSO4 and testing the soluble fraction) has shown galactose oxidase to be consistently very active. However cells expressing galactose oxidase fail to produce detectable amounts of H2O2 in the supernatant. This is true even of JI377 cells (DE katE, katG, ahp) which cannot scavenge hydrogen peroxide. The leading hypothesis for this inactivity is that galactose oxidase doesn't have sufficient access to its cofactor, Cu2+. Cells grown with copper, thoroughly washed, and then lysed show a great reduction in the activity of galactose oxidase. For this reason, we are looking into the alternative oxidase, pyruvate oxidase.

Pyruvate oxidase: Pyruvate oxidase (spxB) from Streptococcus pnumoniea (R6) is being explored for its ability to produce H2O2in vivo. This enzyme is responsible for the hydrogen peroxide that this pathogen naturally produces when it infects the lungs of humans. The most attractive part of this enzyme it that there should be no transport issues for its substrait (pyruvate) into the cell. Pyruvate oxidase is currently being tested in E. coli in a catalase proficient strain (DH10B) and a catalase deficient strain (JI377), either under the control of a strong constitutive promoter (J23100.B0034) or an AHL inducible promoter (F2622.B0034). The first bit of good news is that catalase deficient cells cannot grow when constitutively expressing pyruvate oxidase, while catalse proficient cells can. This suggests the toxicity is mediated by H2O2 rather than high protein loads or the toxicity of pyruvate oxidase.

Cloning Progress

8/5/08

Good by colony PCR: Pyruvate oxidase in front of a double terminator (spxB.B0015)

Successful SDM of katG and katGssrA to remove EcoRI site.

8/11/08

Pyruvate oxidase is under the control of a strong constitive promoter (J23100.B0034) and an AHL inducible promoter (F2622.B0034)